Strains and methods for improving ruminant health and/or performance

ABSTRACT

Described are strains including  Enterococcus faecium  strain 8G-1 (NRRL B-50173),  Enterococcus faecium  strain 8G-73 (NRRL B-50172),  Bacillus pumilus  strain 8G-134 (NRRL B-50174) and strains having all of the identifying characteristics of each of these strains. One or more of the strains can be used to treat or prevent acidosis. They can also be used to improve other measures of ruminant health and/or performance. Methods of using the strains, alone and in combination, are described. Methods of making the strains are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 61/119,256, filed Dec. 2, 2008, theentirety of which is incorporated by reference herein.

BIBLIOGRAPHY

Complete bibliographic citations of the references referred to herein bythe first author's last name and year of publication can be found in theBibliography section, immediately preceding the claims.

FIELD OF THE INVENTION

The invention relates to strains and methods for controlling acidosis.More particularly, the invention relates to bacterial strains useful forimproving ruminant health and/or performance and methods of making andusing the strains.

DESCRIPTION OF THE RELATED ART

The feeding of high concentrations of fermentable carbohydrate toruminants has become a common practice in the beef and dairy cattleindustry over the last 50 years. The need for improving the productionefficiency and quality of meat has led to this trend. Improvements inproduction have not occurred without certain difficulties. Increasingthe ruminant consumption of fermentable carbohydrate by feeding higherlevels of cereal grains has resulted in increased incidence of metabolicdisorders such as acidosis. The relationship between high concentrateconsumption and ruminal acidosis has been well documented in reviews(Dunlop, 1972; Slyter, 1976). Many researchers have shown a decline inruminal pH following the feeding of high levels of readily fermentablecarbohydrate (RFC) to cattle and the subsequent disruption of ruminalmicrobiota and physiological changes occurring in the animal (Allison,et. al., 1975, Hungate et. al., 1952; Elam, 1976). Most have attributedthis decline to an over production of organic acids by ruminal bacteriasuch as Streptococcus bovis. However, the effect of excessivecarbohydrate on the ruminal microbiota that initiates this response hasnot been well documented.

In the past, intensive management of feeding has been the only method tocombat acidosis. More specifically, grains are diluted with roughage andthe increase in dietary concentrate percentage is carefully controlledin a step-wise method to ensure smooth transition to high levels ofconcentrate over a 14-21 day period. Most commercial feedlots formulateand deliver several “adaptation” diets that contain different ratios ofgrain to forage.

Although intensive feeding management is usually quite effective incontrolling acidosis, it is very costly to the producer due to the highcost of producing, transporting, chopping forage, disposing of increasedanimal waste, and lower production efficiencies. Producers and feedlotmanagers need to implement strategies that will allow for efficientproduction of livestock fed high concentrate rations.

Other strategies have been to combine the use of adaptation diets withfeeding antimicrobial components such as ionophores. Ionophores inhibitintake and reduce the production of lactic acid in the rumen by reducingthe ruminal populations of gram-positive, lactic acid-producingorganisms such as Streptococcus bovis and Lactobacillus spp. (Muir etal. 1981).

Although the usage of ionophores have reduced the incidence of acuteacidosis in feedlots, consumer concern about the use of antibiotics inmeat production and the need for feedlot managers to continually findways to reduce costs while improving animal performance and carcasscomposition has lead to the examination of alternative methods to reduceacidosis and improve feedlot cattle performance.

The use of direct-fed microbials as a method to modulate ruminalfunction and improve cattle performance has been gaining increasedacceptance over the past 10 years. There are two basic direct-fedmicrobial technologies that are currently available to the beef industryfor the control of ruminal acidosis: (1) using lactic acid producing DFMtechnology and (2) adding specific bacterial species capable ofutilizing ruminal lactic acid. While the reported mode of action of eachof these technologies is different, they both attempt to address theaccumulation of ruminal lactic acid.

The first approach, i.e., using lactic acid producing DFM technology,attempts to increase the rate of ruminal lactic acid utilization bystimulating the native ruminal microbiota. As reported, the addition ofrelatively slow growing lactic acid producing bacteria, such as speciesof Enterococcus, produces a slightly elevated concentration of ruminallactic acid. The gradual increase forces the adaptation of the ruminalmicroflora to a higher portion of acid tolerant lactic acid utilizers.However, these Enterococcus strains failed to adequately control andprevent acidosis.

The second approach, i.e., adding specific bacterial species capable ofutilizing ruminal lactic acid, is based on the finding that species ofPropionibacterium significantly minimize the accumulation of ruminallactic acid during an acidosis challenge with a large amount of ReadilyFermentable Carbohydrate (RFC). Propionibacterium are naturalinhabitants of the rumen in both dairy and beef cattle and function inthe rumen by using lactic acid to produce important volatile fatty acidslike acetate and propionate.

Current DFM technologies developed to date have been developed basedupon an antiquated microbiological understanding of the incidence ofacidosis in the rumen. Until recently, methods of studying the microbialecology of the rumen have relied on cultivation techniques. Thesetechniques have been limited due to unknown growth requirements andunsuitable anaerobic conditions for many of the rumen microorganisms.Thus, ecological studies relying on these cultivation techniques havebeen based on a limited understanding of the rumen microbiota.

Current DFMs when used alone or with yeast to minimize the risk ofruminal acidosis and to improve utilization of a feedlot cattle dietcontaining high concentrate provide mixed results. However, a study ofDFM strains Propionibacterium P15, and Enterococcus faecium EF212, andE. faecium EF212, fed alone or fed combined with a yeast, Saccharomycescerevisiae, indicated that addition of DFM combined with or withoutyeast had no effect on preventing ruminal acidosis (Yang, W., 2004).

In view of the foregoing, it would be desirable to provide one or morestrains to prevent and/or treat acidosis. It would be advantageous ifthe one or more strains also improved other measures of ruminant healthand/or performance. It would also be desirable to provide methods ofmaking and using the strains.

SUMMARY OF THE INVENTION

The invention, which is defined by the claims set out at the end of thisdisclosure, is intended to solve at least some of the problems notedabove. Isolated strains are provided, including Enterococcus faeciumstrain 8G-1 (NRRL B-50173), a strain having all of the identifyingcharacteristics of Enterococcus faecium strain 8G-1 (NRRL B-50173),Enterococcus faecium strain 8G-73 (NRRL B-50172), a strain having all ofthe identifying characteristics of Enterococcus faecium strain 8G-73(NRRL B-50172), Bacillus pumilus strain 8G-134 (NRRL B-50174), a strainhaving all of the identifying characteristics of Bacillus pumilus strain8G-134 (NRRL B-50174), and combinations thereof.

Additionally provided is a combination including one or more of thestrains listed above and monensin.

Also provided is a method of administering an effective amount of one ormore of the strains listed above to an animal and a method ofadministering a combination including an effective amount of one or moreof the strains listed above and monensin to an animal.

In at least some embodiments, the administration of the one or morestrain to the animal provides at least one of the following benefits inor to the animal when compared to an animal not administered the strain:(a) reduces acidosis, (b) stabilizes ruminal metabolism as indicated bydelayed lactic acid accumulation and prolonged production of volatilefatty acids, (c) recovers more quickly from acidosis challenge asmeasured by pH recovery and lactic acid decline, (d) reduces exhibitionof clinical signs associated with acidosis (e) increased milk productionin lactating dairy cows, (f) increased milk fat content in lactatingdairy cows, (g) decreased somatic cell count (SCC) in lactating dairycows, (h) improved immunological response and health as evidenced bydecreased SCC and (i) increased efficiency of milk production inlactating dairy cows.

Also provided is a method of making a direct-fed microbial. In themethod, a strain selected from the group consisting of Enterococcusfaecium strain 8G-1 (NRRL B-50173), Enterococcus faecium strain 8G-73(NRRL B-50172), and Bacillus pumilus strain 8G-134 (NRRL B-50174) isgrown in a liquid nutrient broth. The strain is separated from theliquid nutrient broth to make the direct-fed microbial. In at least someembodiments of the method, the strain is freeze dried.

Additionally provided is a method of making a direct-fed microbial. Inthe method, a strain selected from the group consisting of Enterococcusfaecium strain 8G-1 (NRRL B-50173), Enterococcus faecium strain 8G-73(NRRL B-50172), and Bacillus pumilus strain 8G-134 (NRRL B-50174) isgrown in a liquid nutrient broth. The strain is separated from theliquid nutrient broth to make the direct-fed microbial. Monensin isadded to the direct-fed microbial.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred exemplary embodiments described herein are illustrated in theaccompanying drawings, in which like reference numerals represent likeparts throughout and in which:

FIG. 1 is a graph showing pH differences between tester (non-acidotic;Cluster 2) and driver (acidotic; Cluster 1) populations.

FIG. 2 is a graph showing lactic acid accumulation differences betweentester (non-acidotic; Cluster 2) and driver (acidotic; Cluster 1)populations.

FIG. 3 is a graph showing in vitro glucose by treatment over time.

FIG. 4 is a graph showing in vitro lactic acid accumulation by treatmentover time.

FIG. 5 is a graph showing total VFA (acetate+propionate+butyrate)accumulation over time.

FIG. 6 is a graph showing mean ruminal pH over time in control andcandidate DFM cattle.

FIG. 7 is a graph showing mean ruminal lactate over time in control andcandidate DFM cattle.

FIG. 8 is a graph showing ruminal VFA concentrations over timetreatment. (Total VFA=acetate+propionate+butyrate).

Before explaining embodiments described herein in detail, it is to beunderstood that the invention is not limited in its application to thedetails of construction and the arrangement of the components set forthin the following description or illustrated in the drawings. Theinvention is capable of other embodiments or being practiced or carriedout in various ways. Also, it is to be understood that the phraseologyand terminology employed herein is for the purpose of description andshould not be regarded as limiting.

DETAILED DESCRIPTION

Provided herein are strains. Methods of making and using the strains arealso provided.

In at least some embodiments, a direct-fed microbial (DFM) made with oneor more of the strains provided herein allows beef and dairy producersto continue managing feeding regimens to optimize growth and performancewithout sacrificing health due to digestive upset associated withruminal acidosis. At least some embodiments of the DFMs were selected onthe basis of managing ruminal lactate concentrations via lactateutilization or priming the rumen to maintain lactate utilizingmicroflora. At least some embodiments of the DFMs were developed tomanage ruminal energy concentrations. Unlike the current DFMs marketedto cattle producers to alleviate acidosis, at least some of theembodiments of the invention were not developed to manage a problemafter it occurs, but rather to alleviate the problem before it happens.

Strains:

The strains provided herein include Enterococcus faecium strain 8G-1,Enterococcus faecium strain 8G-73, and Bacillus pumilus strain 8G-134,which are also referred to herein as 8G-1, 8G-73, and 8G-134,respectively.

Strains Enterococcus faecium strain 8G-1, Enterococcus faecium strain8G-73, and Bacillus pumilus strain 8G-134 were deposited on Aug. 29,2008 at the Agricultural Research Service Culture Collection (NRRL),1815 North University Street, Peoria, Ill., 61604 and given accessionnumbers B-50173, B-50172, and B-50174, respectively. The deposits weremade under the provisions of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. One or more strain provided herein can be used as adirect-fed microbial (DFM).

For purposes of this disclosure, a “biologically pure strain” means astrain containing no other bacterial strains in quantities sufficient tointerfere with replication of the strain or to be detectable by normalbacteriological techniques. “Isolated” when used in connection with theorganisms and cultures described herein includes not only a biologicallypure strain, but also any culture of organisms which is grown ormaintained other than as it is found in nature. In some embodiments, thestrains are mutants, variants, or derivatives of strains 8G-1, 8G-73, or8G-134 that also provide benefits comparable to that provided by 8G-1,8G-73, and 8G-134. In some embodiments, the strains are strains havingall of the identifying characteristics of strains 8G-1, 8G-73, or8G-134. Further, each individual strain (8G-1, 8G-73, or 8G-134) or anycombination of these strains can also provide one or more of thebenefits described herein. It will also be clear that addition of othermicrobial strains, carriers, additives, enzymes, yeast, or the like willalso provide control of acidosis and will not constitute a substantiallydifferent DFM.

Bacillus strains have many qualities that make them useful as DFMs. Forexample, several Bacillus species also have GRAS status, i.e., they aregenerally recognized as safe by the US Food and Drug Administration andare also approved for use in animal feed by the Association of AmericanFeed Control Officials (AAFCO). The Bacillus strains described hereinare aerobic and facultative sporeformers and thus, are stable. Bacillusspecies are the only sporeformers that are considered GRAS. A Bacillusstrain found to prevent or treat acidosis is Bacillus pumilus strain8G-134.

Enterococcus strains also have many qualities that make them useful asDFMs. Enterococcus strains are known to inhabit the gastrointestinaltract of monogastrics and ruminants and would be suited to survive inthis environment. Enterococcus have been shown to be facultativelyanaerobic organisms, making them stable and active under both aerobicand anoxic conditions. Enterococcus faecium strain 8G-1 and Enterococcusfaecium strain 8G-73 were identified by the inventors as being usefulfor these purposes.

Preparation of the Strains:

In at least one embodiment, each one of the strains described herein iscultured individually using conventional liquid or solid fermentationtechniques. In at least one embodiment, the Bacillus strain andEnterococcus strains are grown in a liquid nutrient broth, in the caseof the Bacillus, to a level at which the highest number of spores areformed. The Bacillus strain is produced by fermenting the bacterialstrain, which can be started by scaling-up a seed culture. This involvesrepeatedly and aseptically transferring the culture to a larger andlarger volume to serve as the inoculum for the fermentation, which canbe carried out in large stainless steel fermentors in medium containingproteins, carbohydrates, and minerals necessary for optimal growth.Non-limiting exemplary media are MRS or TSB. However, other media canalso be used. After the inoculum is added to the fermentation vessel,the temperature and agitation are controlled to allow maximum growth. Inone embodiment, the strains are grown at 32° to 37° under agitation.Once the culture reaches a maximum population density, the culture isharvested by separating the cells from the fermentation medium. This iscommonly done by centrifugation.

In one embodiment, to prepare the Bacillus strain, the Bacillus strainis fermented to a 5×10⁸ CFU/ml to about 4×10⁹ CFU/ml level. In at leastone embodiment, a level of 2×10⁹ CFU/ml is used. The bacteria areharvested by centrifugation, and the supernatant is removed. Thepelleted bacteria can then be used to produce a DFM. In at least comeembodiments, the pelleted bacteria are freeze-dried and then used toform a DFM. However, it is not necessary to freeze-dry the Bacillusbefore using them. The strains can also be used with or withoutpreservatives, and in concentrated, unconcentrated, or diluted form.

The count of the culture can then be determined. CFU or colony formingunit is the viable cell count of a sample resulting from standardmicrobiological plating methods. The term is derived from the fact thata single cell when plated on appropriate medium will grow and become aviable colony in the agar medium. Since multiple cells may give rise toone visible colony, the term colony forming unit is a more useful unitmeasurement than cell number.

Using the Strains:

In at least some embodiments, one or more strain is used to form a DFM.One or more carriers, including, but not limited to, sucrose,maltodextrin, limestone, and rice hulls, can be added to the strain.

To mix the strain(s) and carriers (where used), they can be added to aribbon or paddle mixer and mixed preferably for about 15 minutes,although the timing can be increased or decreased. The components areblended such that a uniform mixture of the cultures and carriers result.The final product is preferably a dry, flowable powder, and may beformulated based upon the desired final DFM concentration in the endproduct.

In at least one embodiment of a method of making a DFM, a straindescribed herein is grown in a medium, such as a liquid nutrient broth.The strain is separated from the liquid nutrient broth to make thedirect-fed microbial. The strain can be freeze dried after it isseparated from the broth.

One or more of Enterococcus faecium strain 8G-1, Enterococcus faeciumstrain 8G-73, and Bacillus pumilus strain 8G-134 can be fed to animalsto reduce or even eliminate the occurrence of acidosis. For this, aneffective amount of one or more of these strains is administered to theanimals. Upon administration to the animals, the strain(s) provides atleast one of the following benefits in or to the animals: (a) reducesacidosis in the animals, (b) stabilizes ruminal metabolism as indicatedby delayed lactic acid accumulation and prolonged production of volatilefatty acids, (c) recovers more quickly from acidosis challenge asmeasured by pH recovery and lactic acid decline, and (d) does notexhibit clinical signs associated with acidosis.

The animals can be cattle, including both beef cattle and dairy cattle,that is, one or more bull, steer, heifer, calf, or cow; goats; sheep;llamas; alpacas; other four-compartment stomached, and ruminant animalsthat may encounter ruminal imbalance when fed readily fermentablecarbohydrate (RFC).

In at least one embodiment, when Enterococcus faecium strain 8G-1 orEnterococcus faecium strain 8G-73 is fed, the strain is administered tothe animals at a level such that the animals are dosed daily with about5×10⁸ CFU/animal/day to about 5×10¹⁰ CFU/animal/day. In at least oneembodiment, when Bacillus pumilus strain 8G-134 is fed, the strain isadministered to the animals at a level such that the animals are doseddaily with about 5×10⁸ CFU/animal/day to about 5×10¹⁰ CFU/animal/day. Inat least one embodiment, two or more strains of Enterococcus faeciumstrain 8G-1, Enterococcus faecium strain 8G-73 and Bacillus pumilusstrain 8G-134 are fed, and the strains are administered to the animalsat a level such that the animals are dosed daily with about 5×10⁸CFU/animal/day to about 5×10¹⁰ CFU/animal/day as the total dose of thecombined strains. Other levels of one or more strains can be fed to theanimals.

The strain can be administered to the animals from about 30 days of agethrough the remainder of the adult ruminant productive life or for othertime periods.

In at least one embodiment, the strain is fed as a direct-fed microbial(DFM), and the DFM is used as a top dressing on a daily ration. Inaddition, the strain can be fed in a total mixed ration, pelletedfeedstuff, mixed in with liquid feed, mixed in a protein premix,delivered via a vitamin and mineral premix.

In at least one embodiment, the strain is fed as a DFM, and the DFM isfed in combination with Type A Medicated Article monensin (Rumensin®),with a daily dose about 50 mg to 660 mg per head Monensin is fed toincrease feed efficiency. Monensin, as an ionophore, createspermeability in bacterial cell membrane creating an ion imbalancebetween the intracellular and extracellular spaces. This responseaffects ruminal microbiota populations and influence feedstufffermentation to improve livestock feed efficiency.

In at least one embodiment, the strain is fed as a DFM, and the DFM isfed in combination with Type A Medicated Article tylosin phosphate(Tylan®), with a daily dose of about 60 to 90 mg/head. Tylosin phosphateis fed to beef cattle to reduce liver abscesses caused by Fusobacteriumnecrophorum and Actinomyces pyogenes.

Examples

The following Examples are provided for illustrative purposes only. TheExamples are included herein solely to aid in a more completeunderstanding of the presently described invention. The Examples do notlimit the scope described herein described or claimed herein in anyfashion.

Example 1 Acidosis Model Experimental Design:

Ten crossbred steers were blocked by weight and assigned to two pens.The daily feed ration for all treatment groups prior to challengeconsisted of 45% roughage and 55% concentrate on a dry matter basis.Cattle were fed 15 lbs/head/day of the ration once in the morning andhad remaining feed pushed closer to the feeding stanchion late in theafternoon. Both pens were fasted for 24 hours before challenge with theconcentrate diet treatments. Concentrate diet treatments consisted ofhighly fermentable carbohydrate sources of steam flaked corn on a 90% asfed basis. After fasting for 24 hours, the concentrate diet was fed adlibitum at 100 lbs/pen to all pens (0 h). Challenge diet consumption wasvisually monitored and additional feed added on an as needed basis.

Rumen fluid samples were obtained from individual animals via oralintubation using a collection tube attached to a vacuum flask. Differentflasks and collection tubes were used for each pen to minimize crosscontamination of microbiota between treatments. Ruminal fluid collectedin the vacuum flasks was decanted into sterile 50 ml Falcon tubeslabeled with sample time and animal identification number (ear tagnumber). Ruminal samples were collected from all pens at −36 h, −24 h,and −12 h. Time −36 h and −24 h samples represented the physiologicalbaseline for each animal. Time −12 h samples represented rumen fluid inthe fasted state for each animal. Time 0 h was designated as thebeginning of the feeding challenge. Ruminal samples were collected fromall animals every 4 hours from +6 to +22 hours. All pens were sampled at+28, +36, and +48 hours. The pH from individual ruminal samples wereanalyzed immediately after acquisition. All samples were frozen andprepared for shipment to Agtech Products, Inc. (Waukesha, Wis.) forfurther analysis.

Volatile fatty acids and carbohydrate concentrations were measured inindividual ruminal samples. Samples were prepared for HPLC analysis byaseptically removing duplicate 1.0 ml samples from the rumen fluidcollected from each animal at each time period. Samples were placed in a1.5 ml microcentrifuge tube and the debris was pelleted bycentrifugation (10 minutes, at 12,500 rpm). The supernatant fluid (750μl) was transferred to a clean tube and acidified with an equal volumeof 5 mM H₂SO₄. The acidified fluid was thoroughly mixed and filteredthrough 0.2 μm filter directly into a 2 ml HPLC autosampler vial andcapped. Samples were analyzed using a Waters 2690 HPLC system (WatersInc., Milford, Mass.). The sample were injected into 5 mM H₂SO₄ mobilephase heated to 65° C. and separated using a BioRad HPX-87H Column(Bio-Rad Laboratories, Inc., Hercules, Calif.). The HPLC wasstandardized using a set of concentrations for each compound ofinterest. Compounds used as standards were include dextrose (glucose),lactate, methylglyoxal, butyrate, propionate, and acetate.

Discovery of Bacterial Genes in Non-Acidotic Cattle Ruminal Microflora:

Suppressive Subtractive Hybridization:

The Genome Subtraction Kit (Clontech, Palo Alto, Calif.) was utilized todetermine microbial population differences between two sets of pooledruminal samples. Hierarchal clustering analysis was performed todetermine similarities and differences between animals based on pH andlactic acid profiles over time. Cluster analysis positioned cattle 2069,2071, 2078, 2113, and 2127 in Cluster 1 and cattle 2107, 2115, 2088,2133, and 2124 in Cluster 2. Repeated measures analysis was performed tocompare pH and lactic acid from Cluster 1 to Cluster 2. All variableswere analyzed separately. Cluster 1 had a significantly higher meanlactic acid profile than Cluster 2 (P=0.0004) accompanied with lowermean pH (P=0.0075) throughout the course of the challenge diet period(FIGS. 1 and 2). The rumen fluid from individual animal was pooledwithin cluster for suppressive subtractive hybridization (SSH)procedures.

Suppressive subtraction hybridization (SSH) strategies were developed tocompare pooled ruminal DNA samples from cattle in Cluster 1 to those inCluster 2 at sample times +6 h, +10 h, +14 h, and +18 h. Suppressivesubtraction hybridization was performed utilizing Cluster 2 as thetester (non-acidotic cattle) and Cluster 1 as the driver (acidoticcattle). The SSH was hypothesized to result in unique DNA fragments fromorganisms that resulted in lower levels of lactic acid and a higher pH(ruminal energy modulating organism). By performing subtractions usingsamples from time +10 h, the DNA fragments (genes) found, were fromorganisms that were able to modulate the utilization of excess energy inthe ruminal environment in the form of RFC and alleviate potentialeffects of acidosis.

Cloning and Screening of Unique Tester Sequence Library:

Strain specific DNA sequences that are recovered after subtraction werecloned for further analysis. DNA sequences were inserted into the pCR2.1vector (Invitrogen) and transformed into E. coli chemically competentTOP10 cells. The transformation mixture was plated onto 22×22 cm LB agarplates containing 50 μg/ml kanamycin and overlaid with 40 mg/ml X-gal inDMF. Plates were incubated at 37° C. for 24 h. Recombinant colonies(white colonies) were picked into sterile microtitre plates containingLB medium and kanamycin at 50 μg/ml. All wells containing recombinantPCR products were separated into 1 ml aliquots. One aliquot was purifiedusing the Qiaquick PCR Purification Kit (Qiagen), with the secondaliquot pelleted via centrifugation, resuspended in LB+Kan+10% glyceroland stored at −80° C.

Southern Hybridization:

Slot-blot hybridizations were conducted using standard protocols. Toconfirm the specificity of the cloned DNA inserts, positively chargedZeta-Probe® Blotting Membranes (Bio-Rad Laboratories; Hercules, Calif.)were hybridized with probes made from the original tester and driver DNAdigested with Alu I and labeled with the DIG High Prime DNA labeling kit(Roche Diagnostics Corporation, Indianapolis, Ind.). Recombinant insertsshowing sequence homology to the tester DNA but not the driver DNA wasselected for sequence analysis. Hybridizations were conducted on clonedinserts. At each time period, subtraction was performed, SSH 6, 10, 14,and 18. From SSH 6, 10, 14, and 18, there were 12, 29, 105, and 29cloned inserts, respectively, that were tester specific.

The DNA sequence from each tester positive insert was determined (LarkTechnologies; Houston, Tex.). Sequence from each insert was comparedwith sequences from the NCBI database using the blastX function.Nucleotide sequences were translated and gene function was deduced bycomparing sequences to those found in the NCBI database using the blastXfunction. Gene function was placed in a gene category using the Clustersof Orthologous Groups (COG) web site. Specific COG genes identified wereused to construct oligonucleotide probes for colony hybridization andslot-blot hybridization experiments. Four genes of the twenty-nine wereselected from SSH 10 to be utilized for colony hybridization based uponfunctional attributes based on selection from non-acidotic cattle. Thegenes were selected from clones 79, 84, 94, and 110 were identified viausing the NCBI blastX function with assigned functions: beta-xylosidase,glucose/galactose transporter, 4-alpha-glucanotransferase, and4-alpha-glucanotransferase, respectively. All genes selected for colonyhybridization had assigned properties as identified by COG asCarbohydrate and Transport Metabolism function, which would haveprovided bacteria containing these genes an advantage at metabolizingexcess energy such as that found in the rumen when challenged with RFC.

Colony Hybridization:

Rumen fluid collected during the acidosis trial from cattle at times +10h, +14 h, and +18 h was utilized. Cattle 2107, 2124, 2115, 2088, and2133 were selected from each of these time periods. These cattle arerepresentative of animals that were previously selected for the “testerpopulation” or non-acidotic group. Individual rumen samples were takenfrom −20° C. and allowed to thaw at room temperature. Thawed rumensamples were individually plated on three separate mediums in duplicate.Media utilized consisted of sodium lactate agar (NLA), LactatePropionibacterium Selective Agar (LPSA), and modified reinforcedClostridial media (RCS). The RCS was prepared similar to commerciallyavailable reinforced Clostridial media sans glucose. Thus, the majorcarbohydrate source in RCS is starch. Table 1 below indicates theincubation conditions and dilutions of rumen fluid plated on each media.

TABLE 1 Incubation conditions and dilutions plated on each media.Incubation Conditions Incubation Dilutions Media O2 ConditionsIncubation Time Temperature Plated LPSA Anaerobic  7 Days 32° C. 10-1,10-2 NLA Anaerobic  5 Days 37° C. 10-2, 10-3 RCS Anaerobic 48 Hours 37°C. 10-1, 10-2

After incubation, individual colonies were picked off of each plate andinoculated into 10 ml broth tubes consisting of the respective media,except LPSA, which was inoculated into NLB. Colonies were selected fromeach time period and each animal (five cattle×three time periods). Forthe RCS media, five colonies were picked for each animal-time period.The LPSA exhibited less colonies and diversity on the plates and numberof colonies selected per animal-time period was variable. Two coloniesper animal-time period were selected from the NLA media, except animal2107 at time period 18. Six colonies were picked from this animal-timeperiod due to increased visible diversity. Not all inoculated tubesexhibited growth after incubation.

Tubes showing growth were separated into two separate aliquots of 9 mland 1 ml. The 1 ml aliquot was utilized for DNA isolation proceduresutilizing the High Pure PCR Template Preparation Kit (Roche MolecularBiochemicals; Mannheim, Germany). The 9 ml aliquot was transferred to asterile 15 ml Falcon Tube and centrifuged until a solid pellet wasformed. The pellet was then reconstituted in NLB or RCS brothscontaining 10% glycerol. The reconstituted sample was placed in the −80°C. for future use. The extracted DNA was then used for RAPD-PCR analysisof individual isolates to determine phylogenic relationships. Analysiswas performed using Bio-Numerics (Applied Maths Inc., Austin, Tex.) onthe RAPD DNA banding patterns to determine the relatedness of theisolates. The similarity coefficient of isolates was determined usingthe Dice coefficient and an un-weighted pair group method (UPGMA). Asimilarity of 80% or greater was used to group the 109 isolates into 65separate clusters. Of the 65 clusters, 23 grew only on RCS, 11 grew onlyon LPSA, 14 grew only on NLA, 4 clusters grew both on RCS and LPSA, 6grew on both RCS and NLA, 3 grew on both LPSA and NLA, and 4 clusterswere found to be present on all three media.

Slot blot hybridizations were prepared utilizing the Bio-Dot SFMicrofiltration Apparatus (BIO-RAD; Hercules, Calif.). The genomic DNAof a single isolate within a cluster was selected to represent thecluster and blotted onto membranes. Probes were prepared forhybridization using the PCR DIG Probe Synthesis Kit (Roche MolecularBiochemicals; Mannheim, Germany). Probes selected were derived from thecloned insert analysis described above and consisted of four cloneinserts (Clones 79, 84, 94, and 110) from SSH10. Labeled probes werepooled prior to hybridizations. Hybridizations were conducted at 45° C.for 5 hours. Colorimetric reactions were allowed to develop overnight onthe membranes. Thirty of the 37 isolates (clusters) on the RCS membraneexhibited hybridization as identified by colorimetric reaction and 25 ofthe 28 isolates on the LPSA/NLA membrane

Isolates exhibiting hybridization were then prepared for 16 s rRNAsequencing. Briefly, the 16 s rRNA of each of the 55 isolates wasamplified via PCR using the primers 8F (AGAGTTTGATYMTGGCTCAG) and 1406R(ACGGGCGGTGTGTRC). The PCR product was purified using the QIAquick PCRpurification kit (Qiagen, Valencia, Calif.). Purified product wasanalyzed by gel electrophoresis. When sufficient product was available,the purified sample was sent overnight on ice for single pass sequencing(Lark Technologies, Houston, Tex.). The 16 s sequences from each clusterwere compared with sequences from the NCBI database using the blastnfunction. Organisms of interest brought forward from this comparisonconsisted of Enterococcus faecium strain 8G-1, Enterococcus faeciumstrain 8G-73, and Bacillus pumilus strain 8G-134.

Example 2 In Vitro Strain Testing:

Rumen fluid was collected for in vitro trials from two yearling Herefordheifers. Heifers were identified by identification tags and werereferred to as 101 and 133. Heifers were fed 6 lbs/head/day of drieddistillers grain (DDGS) and had access to free choice haylage.

The in vitro protocol was followed as closely as possible to decreaseexperimental error between each trial. Briefly, rumen fluid wascollected from each heifer and placed into marked, pre-warmed thermoses.Thermoses were transported to Agtech Products, Inc. for processing.Rumen fluid was added in duplicate to bottles containing McDougall'sBuffer and 3.0% glucose (final concentration after McDougall's Bufferand rumen fluid have been mixed to a volume of 180 ml), which had beentempered to 39° C. Candidate DFM strains, Enterococcus faecium strain8G-1, Enterococcus faecium strain 8G-73, and Bacillus pumilus strain8G-134, were added to designated bottles at 1.0×10⁷ CFU/ml (finalconcentration). The unit of observation was the bottle, and treatmentswere performed in quadruplicate. Treatments consisted of Control(glucose added but no DFM), Enterococcus faecium strain 8G-1,Enterococcus faecium strain 8G-73, and Bacillus pumilus strain 8G-134.Bottles were then purged with of CO₂ and capped. Bottles were maintainedin a shaking water bath at 39° C. and 140 rpm. Approximately 10 minutesprior to sampling, bottles were briefly vented to release gases producedas a byproduct of fermentation. Rumen fluid was withdrawn from eachbottle initially and every 6 hours until the 36 hour mark. Rumen pH andvolatile fatty acids were measured and recorded. Statistical analysiswas performed using repeated measures analysis to determine DFM effectsover time or one-way ANOVA to determine treatment affects at a specificpoints in time.

The focus of the ruminal in vitro experiments was to determine ifcandidate DFM strains, Enterococcus faecium strain 8G-1, Enterococcusfaecium strain 8G-73, and B. pumilus strain 8G-134, could positivelyinfluence ruminal fermentation in an energy excess environment. Excessglucose was added to each ruminal in vitro, to replicate cattleengorgement with a readily fermentable carbohydrate. As shown in FIG. 3,the addition of each of the candidate strains significantly increasedthe utilization of glucose over time (P=0.0001). In comparison to thecontrol (FIG. 4), the influence on lactic acid production over time wasalso significantly impacted by the addition of the candidate DFM to thechallenged in vitro model (P=0.0025). By time point 36 hours, there was17% less lactic acid production in the B. pumilus and 32% less lacticacid accumulation in both the Enterococcus candidates.

Volatile fatty acid analysis was performed via HPLC. Total VFA(acetate+propionate+butyrate) were significantly affected by addition ofthe Enterococcus candidates (P=0.0279) (FIG. 5). The Enterococcuscandidates, 8G-1 and 8G-73, appeared to increase the amount of total VFAproduced over time. There was no significant affect on total VFAproduction when comparing the B. pumilus candidate to that of thecontrol.

The in vitro results indicated that the candidate DFMs 8G-1, 8G-73, and8G-134 positively affected ruminal fermentation by increasing glucoseutilization without a corresponding increase in lactic acid productionin comparison to that of the control treatment. Excess glucose in therumen is typically fermented rapidly with the production lactic acid. Itis the accumulation of lactic acid which drives an acute acidoticresponse. By utilizing glucose without the concomitant production oflactic acid, the candidate DFMs have demonstrated the potential toameliorate the affects of acidosis. The ruminal in vitro model suggestedthat these strains may be able to successfully modulate excess ruminalenergy in cattle fed high amounts of readily fermentable carbohydrates.

Example 3 Candidate DFM Testing in Cattle Fed a Readily FermentableCarbohydrate—an Acute Acidosis Challenge:

Materials and Methods:

Cattle and Pens Assignments:

Twenty cross-bred beef steers were purchased at local sale barns. Cattlewere housed at the research facility for a period of two weeks prior totrial initiation for observation of morbidity or mortality. Cattle wererandomly blocked across treatment by weight. Five head of cattle wereassigned to a pen and pens designated to one of four treatments.Treatments consisted of 3 pens each receiving a different DFM as isdetailed below with the fourth pen receiving no DFM (control). Treatmentassignments can be seen in Table 2 below.

TABLE 2 Treatment assignments by pen. Candidate Minimum Dose Pen ID DFM(TX) (CFU/Head/Day) 16s rRNA Identification 1 None 0 None (Control) 28G-1 5 × 10¹⁰ Enterococcus spp. 3 8G-73 5 × 10¹⁰ Enterococcus spp. 48G-134 5 × 10⁹ Bacillus pumilusThe daily feed ration for all treatment groups prior to challenge,consisted of 62.5% roughage and 30% cracked corn and 7.5% proteinsupplement (Table 3) below. The protein supplement contained monensin(Rumensin®) fed at 375 mg/head/day. The protein supplement alsocontained tylosin phosphate (Tylan®).

TABLE 3 Challenge Ration Composition Ingredient % of Diet (DM)Pre-challenge diet Ground Hay 62.5 Cracked Corn 30 Steakmaker ® K+ 45-257.5 R500 T180* Challenge Diet Steam Flaked Corn 87.4 Alfalfa Pellets 5.1Steakmaker ® K+ 45-25 7.5 R500 T180* *Both rations contain Rumensin andTylan.Cattle were fed 15 lbs/head/day of the ration once in the morning andhad any remaining feed pushed closer to the feeding stanchion late inthe afternoon. Fourteen days prior to fasting, treatment groups were fedcandidate DFMs at the dose designated in Table 2 above as a top dressingon the daily ration.

Bacillus pumilus strain 8G-134 was fed at a minimum of 5×10⁹CFU/Head/Day. The Enterococcus candidates 8G-1 and 8G-73 were fed at5×10¹⁰ CFU/Head/Day.

Candidate DFM Preparation:

Candidate DFM strains, previously selected for the challenge trial, wereEnterococcus spp. 8G-1 and 8G-73; and Bacillus pumilus strain 8G-134.Strains were stored at −80° C. Each culture was inoculated into 10 mlbroth tubes containing MRS (Man, Rogosa and Sharp) or TSB (tryptic soybroth). Broth tubes were incubated for 24 hours at 32° and 37° C. forthe Bacillus and Enterococcus candidates, respectively. Cultures werestruck for isolation on respective agar medium and incubated. Anisolated colony was picked into 10 ml of broth and allowed to grow tomid log phase (18 to 24 h) and transferred into fresh broth (10% vol/voltransfer). Enterococcus candidates were grown at 37° C. in MRS broth.Bacillus was grown at in a shaking incubator at 130 rpm at 32° C. inhorizontal TSB tubes. For the growth of Enterococcus, 2 ml weretransferred into a 250 ml bottle containing 198 ml of broth andincubated for 18 hrs.

The 200 ml of culture was inoculated into a 2 L bottle containing 1.8 Lof broth and allowed to incubate for 18 hr. For the Bacillus candidates,5 ml were transferred into a 250 flask containing 50 ml of TSB and thenwas incubated at 32° C. in a shaking incubator at 130 rpm for 24 hr. The50 ml was used to inoculate a 1L flask containing 600 ml and allowed toincubate for another 24 hours.

The optical density (OD) of the 18 hr culture of Enterococcus candidateswas taken before harvesting the cells. The OD was compared to previousgrowth curves to determine the cfu/ml of culture. Samples were platedfor enumeration and genetic fingerprinting. Quality control was ensuredbetween each fermentation batch via RAPD-PCR analysis. With a targetminimum of 5.0e10 cfu/head/day for Enterococcus candidates, thecalculated amount of culture was dispensed into 250 ml Nalgen centrifugebottle and spun at 4° C. for 10 min at 4500 rpm. Target minimum forBacillus candidate was 5.0e9 cfu/head/day, and a total of 100 ml of theBacillus culture was spun down similar to the Enterococcus. Supernatantwas discarded. The pellet was resuspended in 30 ml of growth mediacontaining 10% glycerol. This amount was transferred to a 50 ml conicaltube. The centrifuge bottles were then rinsed with 10 ml of broth andtransferred to the same conical tube. Samples were labeled with strain,date the candidate was harvested, and fermentation batch number. Platecounts were used to determine the total cfu in each tube. Tubes werecombined to deliver counts of a minimum of 5.0e10 cfu/head/day forEnterococcus candidates and 5.0e9 cfu/head/day for Bacillus candidates.All conical tubes were frozen at −20° C.

Challenge Diet and Rumen Fluid Collection Phase:

Rumen fluid samples were obtained from individual animals via ruminalintubation using a collection tube fitted with a strainer and attachedto a vacuum source through a vacuum flask. The pH was immediatelymeasured after rumen fluid acquisition and samples were frozen to betransported to Agtech Products, Inc. for VFA analysis. Samples werecollected from all cattle at sample times −12 h, +6 h, +10 h, +14 h, +18h, +22 h, +30 h, +36 h, and +48 h, with time 0 h representing theinitiation of the challenge. All feed was removed from the cattle attime −24 h to initiate the fast and encourage cattle to engorge thechallenge ration at time 0.

All pens were fasted for 24 hours before challenge with the concentratediet (Time 0). The concentrate diet consisted of 28 lb flake weightsteam flaked corn (Table 3 above). The challenge ration was fed todeliver 20 lbs/head. Challenge ration consumption was visually monitoredand additional feed added on an as needed basis through the remainder ofthe trial.

Ruminal samples were collected every 4 hours from all cattle from +6 to+22 hours. Each rumen sample pH was analyzed immediately afteracquisition. Rumen fluid was then frozen and transported to AgtechProducts, Inc. for VFA analysis via HPLC. Repeated measures analysis wasperformed on rumen pH, VFAs, and glucose levels using individual animalas the unit of observation. Pairwise comparisons were performed overtime between each candidate DFM treatment pens and the control pen todetermine the candidates' effectiveness to alter ruminal fermentationpatterns.

Results and Discussion:

Twenty head of crossbred beef cattle weighing on average 731.95 lbs wererandomly blocked by weight across treatments such that there were nosignificant differences by weight between treatment groups (Table 4).There were 3 treatment pens and one control pen with five head/pen.Treatment assignment per pen can be seen in Table 2 above.

TABLE 4 Treatment assignments by pen. Average Feed Ave. Pen Weight inlbs Consumption/Steer¹ Pen Treatment N (SD) (% of Body Weight) 1 Control5 731.6 (79.94) 4.8 2 8G-1 5 741.2 (94.03) 3.9 3 8G-73 5 731.6 (70.12)3.7 4 8G-134 5 727.6 (72.71) 3.6 ¹Average feed consumption/steer wascalculated as a percentage of the average steer weight for that pen

Cattle were fed challenge ration at time 0 and rumen fluid was collectedat designated time points to measure ruminal fermentation values. Feedconsumption per pen was monitored and recorded after 24 hours. Averagefeed consumption/steer was calculated as a percentage of the averagesteer weight for that pen (Table 4 above). The control pen (pen 1)appeared to have the highest consumption of feed in comparison to theother treatment groups with the average steer consuming 4.8% of its bodyweight. The lowest challenge ration consumption/pen was in pen 4 withthe average steer eating approximately 3.6% of its body weight. Cattlein all pens on average would have been consuming approximately 5.625 lbsof concentrate/day as part of the pre-challenge ration, which on averagewould have constituted 0.8% of the average steers' body weight. Despitethe pen variation of challenge ration consumption, the difference wasnot greater than the increase in concentrate consumption from thepre-challenge ration and would not be causative in fermentationdifferences between pens.

After 24 hours, feed was removed from the cattle and ground prairie haywas fed ad libitum. Cattle were given free choice hay as a precautionagainst the continually decreasing rumen pHs. The addition of hay wouldstimulate additional cud chewing and help to buffer the rumen. Despitethe addition of hay, the ruminal pH still continued to decline.

Immediately after rumen fluid collection, sample pH was analyzed. Alltreatment groups exhibited a decline in ruminal pH as can be observed inFIG. 6. The pH for the control group achieved nadir at time 30 hours andbegan to gradually climb thereafter. By the last rumen sample collectionthe mean pH for the control pen was still acutely acidotic with a pH of4.94. Acute acidosis is associated with pH that remains below 5.2 andchronic or subacute acidosis characterized by a pH below 5.6 (Owens, et.al., 1998). Mean numerically higher trends appeared for strains 8G-1,8G-73, and 8G-134 in pens 2, 3, and 4 when compared to that of thecontrol from time +22 to +48. This suggests that cattle treated with thecandidate DFMs in these pens recovered more quickly from the acidoticchallenge. Mean pH for the for cattle treated with 8G-1, 8G-73, and8G-134 at time +48 was 5.96, 6.02, and 6.14, respectively, which isgreater than 1.0 pH unit above the control pen. Repeated measuresanalysis of these three strain over time (+6 to +48) did not exhibitsignificant differences when compared to the control pen. However whenpH comparisons of pens treated with 8G-1, 8G-73, and 8G-134 to thecontrol pen from time +22 h to +48 h were performed, differences were orapproached significance (P=0.1562, 0.0965, and 0.0466 for 8G-1, 8G-73,and 8G-134, respectively).

Average lactic acid profiles for all treatment groups are shown in FIG.7. Mean ruminal lactic acid accumulation peaks at 105 mM for the controlpen 30 hours after receiving challenge ration. Candidate DFM strains8G-1, 8G-73, and 8G-134 again exhibited visible mean numeric differencesin lactate accumulation in comparison to that of the control pen. Meanlactic acid accumulation was similar between the control cattle and the8G-1 treated cattle through the first 14 hours of the challenge.Subsequent accumulation levels for the remainder of the trial were muchless in the 8G-1 treated cattle although not significant (P=0.1892).Treatment pens 8G-73 demonstrated decreased levels of lactic acidaccumulation at times 30 and remained lower than the control pen for theremainder of the trial. Candidate strain 8G-134 also showed decreasedlevels of lactic acid starting at +22 h and remained consistently lowerthan the control pen through +48 h.

Individual VFAs were measured and analyzed. Volatile fatty acid (VFA)concentrations increased in the control pen and treatments 8G-73 and8G-134 and peaked at six hours (FIG. 8). After six hours each of thesetreatment pens showed declining levels of total VFA (acetate, propionateand butyrate). There were no significant differences between thesetreatments and the control. Treatment 8G-1, however, exhibited a delayin VFA decline which did not occur until +14 h. Over the course of thetrial there were no significant differences in total VFA concentrationor the individual VFA (consisting of acetate, propionate, or butyrate)levels.

In addition to monitoring and measuring ruminal fermentationcharacteristics over the course of the acidotic trial, cattle wereobserved throughout the trial for visible clinical effects associatedwith acidosis. Early in the acidotic challenge (+0 h to +14 h), theeffects of the challenge diet were minimal. Cattle did not show signs ofdepression and continued to feed on the challenge ration. By +22 hourspost receiving the challenge ration all cattle except those in receivingTreatment 8G-1) were showing signs of soreness, depression, and hadloose, liquid fecal excretion. Cattle in pen 2 were no longer consumingfeed, but did not exhibit clinical symptoms, despite similar havingsimilarly declining pH levels.

Acute ruminal acidosis by definition is the decline in ruminal pH tolevels deleterious not only to rumen function but also livestock health.Acute acidosis is marked by the accumulation of lactic acid and thedecline in VFA production. Proper rumen function is a combination ofmanaging the available energy and nitrogen components available infeedstuffs. When imbalances in ruminal metabolism occur, digestive upsettypically follows and can manifest in the form acidosis. In this trial,strains 8G-1, 8G-73, and 8G-134 enhanced the recovery of rumen functionas indicated by ruminal fermentation parameters.

Cattle fed 8G-1, Enterococcus faecium, on average recovered more quicklyfrom the acidosis challenge as measured by pH recovery and lactic aciddecline. In addition to the measured ruminal fermentation patterns,cattle fed candidate DFM 8G-1 did not exhibit clinical signs associatedwith acidosis.

Candidate strain 80-73, Enterococcus faecium, improved ruminalfermentation through the course of the trial. Mean lactic acid levelswere the lowest for all candidate strains tested at +48 h at 12.54 mM. Acorresponding increase in pH was also associated with the recovery witha final pH of 6.02, which was 1.08 pH units higher than that of thecontrol.

Candidate strain 8G-134, Bacillus pumilus, also enhanced ruminalrecovery during the acidotic challenge. Mean lactic acid levels, incattle fed 8G-134, peaked at 89 mM at time +22 h, while the control pencontinued to increase and peaked at 105 mM at time +30 hours. Meanlactic acid levels had dropped to 57 mM by +30 hours. As with lacticacid accumulation, ruminal pH in cattle fed 8G-134 recovered morequickly than that of the control and was found to be significantlydifferent from +22 to +48 h (P=0.0466).

Example 4

Summary:

Thirty prima and multiparous Holstein cows were blocked by previouslactation and predicted producing ability (PPA) and assigned to one ofthree treatments. Ten cows were assigned per treatment and treatmentsconsisted of a control group (Treatment 1) which received a basal totalmixed ration (TMR), Treatment 2 and Treatment 3 which received basaltotal mixed ration TMR and were fed Bacillus pumilus 8G-134 at 5×10⁹ and1×10¹⁰ CFU/head/day, respectively, from 3 weeks prepartum to 22 weekafter parturition. The primary objective was to determine the effects ofB. pumilus 8G-134 on dairy cow milk production and performance abovecontrol cattle during this time period. The secondary objective was todetermine if there was a dose response associated with feeding B.pumilus 8G-134. The B. pumilus 8G-134 regimens significantly increasedmilk production, milk fat, and decreased somatic cell count. Thesesignificant B. pumilus 8G-134 production effects did not come at theexpense of cow body condition score, body weight, increases in drymatter intake or significantly change blood metabolite profiles, andwould indicate B. pumilus 8G-134 also provided dairy cow efficiencybenefits.

Materials and Methods:

Livestock:

Thirty Holstein cows were randomly assigned to one of three dietarytreatments in a continuous lactation trial from 3 weeks prior toparturition through 22 weeks postpartum. There were no significantdifferences for previous milk yield for second and older cows or forpredicted producing ability (PPA) for first lactation cows for thedifferent treatment groups. The numbers of first and second lactationanimals deviated between the groups, but did not influence overall meanproduction, as lactation was adjusted in the statistical model. Animalswere on study from approximately three weeks prepartum through 22 weekspostpartum.

Nutrition:

Dietary ingredients and formulated composition of total mixed rations(TMRs) are presented in Table 5 below for dry and lactating cows,respectively. The base TMR was the same for each group and differed bytop dress treatment. Each group received a top dress of 8 ounces offinely ground corn to which was added 1 ounce of maltodextrin (Treatment1, control), Bacillus spp at 5×10⁹ CFU/head/day (Treatment 2), andBacillus spp at 1×10¹⁰ CFU/head/day (Treatment 3).

TABLE 5 Formulated composition of the TMR offered to dry and lactatingcows. Dry Period Lactating Period Ingredients, % DM basis Corn Silage52.57 39.14 Ryelage 18.54 Alfalfa haylage — 16.52 Grass hay 14.45 1.77SBM48 5.74 10.77 Blood Meal 4.41 AminoPlus — 6.01 Corn 2.41 21.07 Fat0.65 1.42 Limestone — 1.26 Sodium bicarbonate — 0.84 MagOx 0.65 0.42Salt 0.32 0.53 TMin Vit 0.27 0.26 Composition, % DM CP 16.50 16.64 SP, %CP 32.29 28.58 NDF 41.06 30.65 Starch 19.56 29.82 Sugar 3.19 2.68 NFC33.47 41.87 Fat 3.82 4.15 Ca 0.31 0.87 P 0.29 0.34 Mg 0.54 0.42 K 1.721.33 NeL, mcal/kg 1.60 1.74

Sample and Data Collection:

Daily TMR samples and refusals were collected and composited weekly,weekly composites combined monthly, and monthly samples were analyzedfor dry matter (DM), crude protein (CP), acid detergent fiber boundprotein (ADF-CP), neutral detergent fiber bound protein (NDF-CP),soluble protein (SP), acid detergent fiber (ADF), neutral detergentfiber (NDF), lignin, fat, starch, sugar, ash, calcium (Ca), phosporus(P), magnesium (Mg), potassium (K), sulfur (S), sodium (Na), chlorine(Cl), iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu) byCumberland Valley Analytical Services, Maugansville, Md.

Cows were milked twice a day, and milk volume was recordedelectronically at each milking and am-pm amounts summed for daily total.Once a week milk samples from am and pm milkings were composited foranalysis of content of fat, protein, somatic cells, solids not fat, andmilk urea nitrogen (MUN) by Dairy One milk laboratory in State College,Pa. using a Fossamatic 4000 (FOSS; Eden Prairie, Minn.).

Animals were on study from approximately three weeks prepartum through22 weeks postpartum. Animal weight was estimated by heart girthcircumference on weeks 1, 3, 7, 11, 15 and 18 postpartum. Body conditionwas assessed by two independent observers at the same time as bodyweight was collected.

Blood samples were collected from the coccygeal vein, serum harvested,frozen and analyzed for glucose, beta-hydroxy butyrate (BHB), andnon-esterified fatty acids (NEFA) at weeks 2 and 8 postpartum. Glucoseand BHB were analyzed using an Abbott Precision Xtra™ meter (AbbottDiabetes Care Inc., Alameda, Calif.). A Randox assay kit (Cat. HN 1530,Randox Laboratories, Northern Ireland) was used to measurenon-esterified fatty acid (NEFA) concentration in serum adopted to anenzyme linked immunosorbant assay (ELISA) plate reader at a wave lengthof 550 nm for multiple samples. The Randox kit uses Acyl CoA synthetaseand oxidase to convert NEFA to 2,3-trans-Enoyl-CoA plus peroxide;peroxide plus N-ethyl-N-(2 hydroxy-3-sulphopropyl) m-toluene leads to apurple product, which is the indicator of NEFA concentration in serum.

Statistical Models.

Milk production and content, body weight, and body condition score wereanalyzed using the mixed procedure in SAS statistical software. Cow wasthe repeated subject with the covariance matrix set to type 1correlation structure. Daily milk observations were aggregated by weekpostpartum. The statistical model was as follows:

Y _(i) =u _(i) +TRT _(j)+Lact_(k)+Week₁ +TRT _(j)*Lact_(k) +TRT_(j)*Week₁ +TRT _(j)*Lact_(k)*Week₁ +e _(jklm)

Where

-   -   Y_(i)=least square mean of the production dependent variables;    -   u_(i)=overall mean of the various production variables;    -   TRT_(j)=jth treatment effect, 1, 2, 3;    -   Lact_(k)=kth lactation, 1, 2+;    -   Week₁=1th week, 1 . . . 22;    -   interaction terms (TRTj*Lactk+TRTj*Week1+TRTj*Lactk*Week1)    -   e_(jklm)=error

Monthly samples of TMR and feed refusals for each treatment were testedfor difference using means procedure in SAS.

Blood concentrations of glucose, BHB, and NEFA, were analyzed using thegeneral linear models in SAS statistical software. Class variables werecow, week and treatment. Treatment was nested in cow and was the errorterm for testing treatment significance. Treatment by week interactionwas tested for statistical significance using the residual error.

Results:

Mean TMR composition for dry and lactating TMRs is presented in Table 6over the course of the study. Composition was not different between thetreatment groups.

TABLE 6 Analyzed composition of TMR for dry cows and lactating cowsTreatment, % DM basis Item 1 2 3 SEM Dry TMR N 3 3 3 CP 13.64 13.1713.11 0.14 ADF 29.55 28.37 27.88 0.53 NDF 48.54 48.04 47.14 0.93 Lignin3.64 3.52 3.58 0.09 Starch 18.74 18.19 17.97 0.87 Sugar 6.71 6.15 6.570.23 Ash 8.44 7.56 7.79 0.10 NFC 39.49 36.93 38.20 0.86 Ca 0.49 0.460.47 0.01 P 0.37 0.35 0.35 0.004 Lactating TMR N 9 9 9 CP, % 14.54 14.8114.37 0.10 ADF, % 21.54 21.38 21.64 0.25 NDF, % 33.51 33.21 33.30 0.32Lignin, % 3.27 3.27 3.26 0.06 Starch, % 25.36 26.52 25.48 0.38 Sugar, %6.25 6.15 6.25 0.15 Fat, % 3.69 3.65 3.62 0.04 Ash, % 8.73 8.67 8.600.09 NFC, % 41.22 42.02 41.44 0.34 Ca, % 0.95 0.97 0.96 0.005 P, % 0.360.36 0.36 0.003 standard error means (SEM), NFC, nonfiber carbohydrateNDF, neutral detergent fiber ADF, acid detergent fiber Treatment 1control; Treatment 2, Bacillus pumilus at 8G-134 5 × 10⁹; Treatment 3,Bacillus pumilus 8G-134 at 1 × 10¹⁰.

One control (Treatment 1) cow exhibited abnormal milk production and herdata was removed from the data analysis. The analysis of milk productionwas repeated on 29 cows. Milk production was significantly influenced bytreatment (Table 7 below). Cows fed the Bacillus pumilus 8G-134 at 5×10⁹CFU/head/day (Treatment 2), and Bacillus pumilus 8G-134 at 1×10¹⁰CFU/head/day (Treatment 3) produced significantly more milk than thecows fed the placebo control. Cows on Treatment 2 and 3 producedapproximately 2 kg more milk than treatments 1 (Table 7). There was asignificant interaction with parity. Production increases weresignificant in second parity cows by 5.2 kg, but no significantdifferences in milk production in first parity cows.

TABLE 7 Least square mean milk production in Holstein cows from calvingthrough 22 weeks postpartum fed a microbial additive. Change Milk,relative to kg/d control, Effect Treatment Lactation kg/d sem kg/d semTreatment 1 33.12^(a) 0.65 0.00 0.66 Treatment 2 35.38^(b) 0.60 2.30*0.60 Treatment 3 35.08^(b) 0.61 1.99* 0.61 Lactation 1 31.59^(a) 0.47−0.83 0.47 Lactation 2 36.84^(b) 0.39 3.09* 0.39 Interaction 1 132.44^(a) 1.07 0.00 1.07 2 1 31.70^(a) 0.93 −0.72 0.93 3 1 31.36^(a)0.93 −1.07 0.93 1 2 33.80^(b) 0.74 0.00 0.74 2 2 39.06^(c) 0.76 5.24*0.76 3 2 38.79^(c) 0.79 5.17* 0.79 Means within group with differentsuperscript differ, P < 0.05 Mean change with * differ significantlyfrom 0 Treatment 1 control; Treatment 2, Bacillus pumilus at 8G-134 5 ×10⁹; Treatment 3, Bacillus pumilus 8G-134 at 1 × 10¹⁰.

Milk fat and yield are presented in Table 8 below. Milk fat wassignificantly increased by Treatments 2 and 3 above the control. Yieldresponses followed milk yield with fat yield increased in the Bacilluspumilus 8G-134 fed treatment groups. Bacillus pumilus 8G-134 cattle fortreatment 2 and 3 yielded significantly higher fat percentage above thatof the control with 0.24% and 0.31% higher levels, respectively (Table8). Coupled with the significant increase in milk production, daily fatyield for both treatment 2 and 3 provided significant increases in dailyfat production above that of the control (Table 8).

TABLE 8 Milk fat content by treatment groups. Fat, Effect TreatmentLactation % sem Fat Yield, kg/d sem Treatment 1 3.57^(a) 0.09 1.206^(a)0.042 2 3.81^(b) 0.08 1.351^(b) 0.039 3 3.88^(b) 0.08 1.351^(b) 0.039Lactation 1 3.76 0.06 1.159^(a) 0.030 2 3.78 0.05 1.395^(b) 0.025 Meanswithin column with different superscript differ by P < 0.05 Treatment 1control; Treatment 2, Bacillus pumilus at 8G-134 5 × 10⁹; Treatment 3,Bacillus pumilus 8G-134 at 1 × 10¹⁰.

Log of the linear Somatic cell count (LogSCC) scores were different bytreatment and lactation. The Bacillus pumilus 8G-134 treatments(Treatments 2 and 3) had significantly lower log linear score than thecontrol cows (Table 9 below). Treatments 2 and 3 cows had LogSCC of 4.97and 4.96, respectively compared to those of the control cows at 5.92.Parity two cows had significantly higher log linear score than firstlactation cows. Somatic cell counts are associated with infection aswell as immunological status and health of the lactating dairy cow.Additionally increased SCC is indicative of inflammatory responses toinfection. Decreases in SCC demonstrated here may indicate that cows fedthe Bacillus pumilus 8G-134 are better immunologically to handleinfectious challenge during lactation and maintain udder and cow health.

TABLE 9 Treatment effects on Somatic Cell Count (SCC). Effect TreatmentLactation LogSCC sem Treatment 1 5.92^(a) 0.32 2 4.97^(b) 0.30 34.96^(b) 0.30 Lactation 1 5.27^(a) 0.23 2 5.86^(b) 0.19 LogScc = log ofsomatic cell count Treatment 1 control; Treatment 2, Bacillus pumilus at8G-134 5 × 10⁹; Treatment 3, Bacillus pumilus 8G-134 at 1 × 10¹⁰.

Data for mean DMI for groups for dry and lactating periods in Table 10below. Dry matter intake for dry cows ranged from 10.79 to 11.64 kg/dacross the groups. Lactating groups consumed 22.02, 21.31 and 21.48 kg/dfor treatment groups 1, 2, and 3, respectively (Table 10). Predicted DMIbased on the NRC equation for cows by week postpartum is presented inTable 10. Intake for treatment 1 was 0.83 kg higher than predicted;intake for treatment 2 was −1.37 kg lower than predicted; intake fortreatment 3 was −1.04 kg lower than predicted. The increase in milkproduction on the Treatments 2 and 3 was accomplished with no increasein DMI. In fact the predicted or expected DMI based on NRC predictionscompared to the group mean intake suggests these cows consumed 1.0 to1.5 kg/d less DMI. Thus, the efficiency of DM utilization was increasedon Treatments 2 and 3.

TABLE 10 Least square means for group feed intake, serum glucose,beta-hydroxy butyrate, non-esterified fatty acids, by treatment group.Treatment group Item 1 sem 2 sem 3 sem Dry Matter Intake, kg/d Dry cows10.79 0.27 11.64 0.25 11.12 0.25 Lactating cows 22.02 0.11 21.31 0.1121.48 0.11 Predicted DMI, kg/d 21.19 0.58 22.68 0.57 22.52 0.58 Serumvalues Glucose, mg/dl 53.95 1.98 51.5 1.98 53.5 1.98 Beta-OH butyrate,mg/dl 1.05 0.15 0.88 0.15 1.15 0.15 NEFA, ueq/ml 0.23 0.04 0.16 0.040.17 0.04 Serum values by week, 2, 8; Glucose, mg/dl, 52.90 2.80 47.002.80 48.90 2.80 55.00 2.80 56.00 2.80 58.10 2.80 BHB, mg/dl 0.92 0.210.86 0.21 1.39 0.21 1.17 0.21 0.89 0.21 0.91 0.21 NEFA, ueq/ml 0.41 0.050.23 0.05 0.28 0.05 0.07 0.05 0.10 0.05 0.07 0.05 BHB = beta-hydroxybutyrate Treatment 1 control; Treatment 2, Bacillus pumilus at 8G-134 5× 10⁹; Treatment 3, Bacillus pumilus 8G-134 at 1 × 10¹⁰. Predicted DMI =(.372 * FCM + 0.0968 * BWT(kg){circumflex over ( )}.75) * (1 −exp(−0.192 * (Week + 3.67)))

The DMI differences and production values could suggest that cows onTreatments 2 and 3 could have mobilized more body tissue than thecontrol cows to produce more milk and eat less than expected. However,serum NEFA, glucose, and BHB suggest these cows were in similar energystatus as control cows (Table 10 above). Additionally, body weight andBCS were similar for the Bacillus groups relative to the control group(Table 11 below). This suggests they did not mobilize more body tissueto produce the additional milk volume, indicating feed conversionefficiency in cows feed Treatments 2 and 3 regardless of dose.

TABLE 11 Least square mean body weight (lb) and body condition score bytreatment groups. Body condition score is the average score of twoobservers. Wt, Effect Treatment Lactation (lb) sem BCS sem Treatment 11343.10 28.49 2.92 0.03 2 1324.60 27.54 3.13 0.03 3 1388.02 27.73 3.060.03 Lactation 1 1223.31 21.87 3.04 0.02 Lactation 2 1476.62 17.94 3.010.02 Interaction Treatment × lactation 1 1 1170.78 27.72 3.06 0.05 2 11242.64 25.26 3.17 0.05 3 1 1208.50 24.00 2.94 0.05 1 2 1486.02 19.332.78 0.04 2 2 1406.42 19.96 3.10 0.04 3 2 1547.85 20.79 3.19 0.04 Wt =weight, lbs BCS = body condition score, scale 1 to 5 by 0.25 points; 1 =emaciated, 2 = thin, 3 = average, 4 = fat, 5 = obese Treatment 1control; Treatment 2, Bacillus pumilus at 8G-134 5 × 10⁹; Treatment 3,Bacillus pumilus 8G-134 at 1 × 10¹⁰.

It is understood that the various preferred embodiments are shown anddescribed above to illustrate different possible features describedherein and the varying ways in which these features may be combined.Apart from combining the different features of the above embodiments invarying ways, other modifications are also considered to be within thescope described herein. The invention is not intended to be limited tothe preferred embodiments described above.

BIBLIOGRAPHY

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Dunlop, R. H. 1972. Pathogenesis of ruminant lactic acidosis. Adv. VetSci. Comp Med. 16:259.

Elam, C. J. 1976. Acidosis in feedlot cattle: Practical observations. J.Anim. Sci. 43:898.

Hungate, R. E., R. W. Dougherty, M. P. Bryant, and R. M. Cello. 1952.Microbiological and physiological changes associated with acuteindigestion in sheep. Cornell Vet. 42:423.

Muir, L. A., E. L. Rickes, P. F. Duquette, and G. E. Smith. 1981.Prevention of induced lactic acidosis in cattle by thiopeptin. J. Anim.Sci. 52:635.

Owens, F. N., Secrist, D. S., Hill, W. J., Gill, D. R. 1998. Acidosis incattle: a review. J. Anim. Sci. 76:275-286.

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Yang, W., 2004. Effects of direct-fed microbial supplementation onruminal acidosis, digestibility, and bacterial protein synthesis incontinuous culture. Animal Feed Science and Technology, 114(4): 179-193.

1. An isolated strain selected from the group consisting of Enterococcusfaecium strain 8G-1 (NRRL B-50173), a strain having all of theidentifying characteristics of Enterococcus faecium strain 8G-1 (NRRLB-50173), Enterococcus faecium strain 8G-73 (NRRL B-50172), a strainhaving all of the identifying characteristics of Enterococcus faeciumstrain 8G-73 (NRRL B-50172), Bacillus pumilus strain 8G-134 (NRRLB-50174), a strain having all of the identifying characteristics ofBacillus pumilus strain 8G-134 (NRRL B-50174), and combinations thereof.2. The strain of claim 1, wherein the strain is Enterococcus faeciumstrain 8G-1 (NRRL B-50173).
 3. The strain of claim 1, wherein the strainis a strain having all of the identifying characteristics ofEnterococcus faecium strain 8G-1 (NRRL B-50173).
 4. The strain of claim1, wherein the strain is Enterococcus faecium strain 8G-73 (NRRLB-50172).
 5. The strain of claim 1, wherein the strain is a strainhaving all of the identifying characteristics of Enterococcus faeciumstrain 8G-73 (NRRL B-50172).
 6. The strain of claim 1, wherein thestrain is Bacillus pumilus strain 8G-134 (NRRL B-50174).
 7. The strainof claim 1, wherein the strain is a strain having all of the identifyingcharacteristics of Bacillus pumilus strain 8G-134 (NRRL B-50174).
 8. Acombination comprising: an isolated strain selected from the groupconsisting of Enterococcus faecium strain 8G-1 (NRRL B-50173), a strainhaving all of the identifying characteristics of Enterococcus faeciumstrain 8G-1 (NRRL B-50173), Enterococcus faecium strain 8G-73 (NRRLB-50172), a strain having all of the identifying characteristics ofEnterococcus faecium strain 8G-73 (NRRL B-50172), Bacillus pumilusstrain 8G-134 (NRRL B-50174), a strain having all of the identifyingcharacteristics of Bacillus pumilus strain 8G-134 (NRRL B-50174), andcombinations thereof; and monensin.
 9. A method comprising administeringto an animal an effective amount of a strain selected from the groupconsisting of a strain selected from the group consisting ofEnterococcus faecium strain 8G-1 (NRRL B-50173), a strain having all ofthe identifying characteristics of Enterococcus faecium strain 8G-1(NRRL B-50173), Enterococcus faecium strain 8G-73 (NRRL B-50172), astrain having all of the identifying characteristics of Enterococcusfaecium strain 8G-73 (NRRL B-50172), Bacillus pumilus strain 8G-134(NRRL B-50174), a strain having all of the identifying characteristicsof Bacillus pumilus strain 8G-134 (NRRL B-50174), and combinationsthereof.
 10. The method of claim 9, wherein upon administration to theanimal, the strain provides at least one of the following benefits in orto the animal when compared to an animal not administered the strain:(a) reduces acidosis, (b) stabilizes ruminal metabolism as indicated bydelayed lactic acid accumulation and prolonged production of volatilefatty acids, (c) recovers more quickly from acidosis challenge asmeasured by pH recovery and lactic acid decline, and (d) reducesexhibition of clinical signs associated with acidosis, (e) increasesmilk production, (f) increases milk fat content, (g) decreases somaticcell count (SCC), (h) improves immunological response and health asevidenced by decreased SCC, and (i) increases efficiency of milkproduction.
 11. The method of claim 9, wherein the animal is a ruminant.12. The method of claim 9, wherein the animal is a bull, steer, heifer,cow or calf.
 13. The method of claim 9, wherein the strain isEnterococcus faecium strain 8G-1 (NRRL B-50173), Enterococcus faeciumstrain 8G-73 (NRRL B-50172), or combinations thereof, and wherein thestrain is administered to the animal at a level such that the animal isdosed daily with about 5×10⁸ CFU/animal/day to about 5×10¹⁰CFU/animal/day.
 14. The method of claim 9, wherein the strain isBacillus pumilus strain 8G-134 (NRRL B-50174), and wherein the strain isadministered to the animal at a level such that the animal are doseddaily with about 5×10⁸ CFU/animal/day to about 5×10¹⁰ CFU/animal/day.15. The method of claim 11, wherein the strain is administered to theanimal starting from about 30 days of age.
 16. The method of claim 9,wherein the animal is a beef animal.
 17. The method of claim 9, whereinthe animal is a dairy cow.
 18. The method of claim 17, wherein uponadministration to the dairy cow, the strain provides at least one of thefollowing benefits in or to the dairy cow when compared to a dairy cownot administered the strain: (a) the strain increases daily fat yieldand milk fat percent in the dairy cow administered the strain, (b) thestrain lowers the log Somatic cell count (LogSCC) scores in the dairycow administered the strain, (c) improves immunological response andhealth as evidenced by decreased LogSCC, (d) increases efficiency ofmilk production in dairy cow administered the strain, and (a) when thedairy cows are second parity or greater cows, the strain increases milkproduction of milk in the dairy cow administered the strain.
 19. Themethod of claim 18, wherein the increase in milk production in the dairycow administered the strain is achieved with substantially no increasein dry matter intake in the dairy cow administered the strain.
 20. Themethod of claim 9, wherein the strain is Enterococcus faecium strain8G-1 (NRRL B-50173) or a strain having all of the identifyingcharacteristics of Enterococcus faecium strain 8G-1 (NRRL B-50173). 21.The method of claim 9, wherein the strain is Enterococcus faecium strain8G-73 (NRRL B-50172) or a strain having all of the identifyingcharacteristics of Enterococcus faecium strain 8G-73 (NRRL B-50172). 22.The method of claim 9, wherein the strain is Bacillus pumilus strain8G-134 (NRRL B-50174) or a strain having all of the identifyingcharacteristics of Bacillus pumilus strain 8G-134 (NRRL B-50174).
 23. Amethod comprising administering to an animal a combination comprising:an effective amount of a strain selected from the group consisting ofEnterococcus faecium strain 8G-1 (NRRL B-50173), a strain having all ofthe identifying characteristics of Enterococcus faecium strain 8G-1(NRRL B-50173), Enterococcus faecium strain 8G-73 (NRRL B-50172), astrain having all of the identifying characteristics of Enterococcusfaecium strain 8G-73 (NRRL B-50172), Bacillus pumilus strain 8G-134(NRRL B-50174), a strain having all of the identifying characteristicsof Bacillus pumilus strain 8G-134 (NRRL B-50174), and combinationsthereof; and monensin.
 24. A method of making a direct-fed microbial,the method comprising: (a) growing in a liquid nutrient broth a strainselected from the group consisting of Enterococcus faecium strain 8G-1(NRRL B-50173), Enterococcus faecium strain 8G-73 (NRRL B-50172),Bacillus pumilus strain 8G-134 (NRRL B-50174), a strain having all ofthe identifying characteristics of Enterococcus faecium strain 8G-1(NRRL B-50173), a strain having all of the identifying characteristicsof Enterococcus faecium strain 8G-73 (NRRL B-50172), and a strain havingall of the identifying characteristics of Bacillus pumilus strain 8G-134(NRRL B-50174); and (b) separating the strain from the liquid nutrientbroth to make the direct-fed microbial.
 25. The method of claim 24,further comprising freeze drying the strain.
 26. The method of claim 24,wherein the strain is Enterococcus faecium strain 8G-1 (NRRL B-50173) ora strain having all of the identifying characteristics of Enterococcusfaecium strain 8G-1 (NRRL B-50173).
 27. The method of claim 24, whereinthe strain is Enterococcus faecium strain 8G-73 (NRRL B-50172) or astrain having all of the identifying characteristics of Enterococcusfaecium strain 8G-73 (NRRL B-50172).
 28. The method of claim 24, whereinthe strain is Bacillus pumilus strain 8G-134 (NRRL B-50174) or a strainhaving all of the identifying characteristics of Bacillus pumilus strain8G-134 (NRRL B-50174).
 29. A method of making a direct-fed microbial,the method comprising: (a) growing in a liquid nutrient broth a strainselected from the group consisting of Enterococcus faecium strain 8G-1(NRRL B-50173), Enterococcus faecium strain 8G-73 (NRRL B-50172),Bacillus pumilus strain 8G-134 (NRRL B-50174), a strain having all ofthe identifying characteristics of Enterococcus faecium strain 8G-1(NRRL B-50173), a strain having all of the identifying characteristicsof Enterococcus faecium strain 8G-73 (NRRL B-50172), and a strain havingall of the identifying characteristics of Bacillus pumilus strain 8G-134(NRRL B-50174); (b) separating the strain from the liquid nutrient brothto make the direct-fed microbial; and (c) adding monensin to thedirect-fed microbial.